EPF-Lausanne/7 September 2009
From 2009.igem.org
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: y = 10a / (a+b) | : y = 10a / (a+b) | ||
: x = 10 - y | : x = 10 - y | ||
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With : a = concentration of RO1 in ng/ul | With : a = concentration of RO1 in ng/ul | ||
Revision as of 07:22, 21 September 2009
Wet Lab
As we got our readout1 (confirmed by PCR on miniprep + digestion assay), we have done the following test : all in LB (while waiting for a better option)
- -Our 3 "right" clones of readout 2, induced by trp, Atc and nothing
- -Our 3 readout 1 (confirmed) induced by trp and nothing
- -A control of the "evolution of fluorescence over time" using the LacI inducible RFP from the registry that we have. This, at least, won't have any interferences.
We did a PCR for inclusion of the BB (LovTAP construct) into a vector, and a purification of the PCR products. The concentrations obtained are acceptable. All were then digested with EcoRI and SpeI.
Culture
The following culture have been made in 25mL LB + corresponding antibiotic, the bacteria being taken from the glycerol stock. They will be tested at Sebastian's Lab this afternoon.
- RO2: clones #4-5-10 (Amp)
- RO1: clones #1-2-3 (Amp)
- LacI-RFP: clones #1-2 (Chl)
After incubation at 37°C, the OD is still at 0.00. At 5:00pm, the OD is at 0.01 -> wait until tomorrow.
Finally we killed them and we will make another overnight culture to inoculate tomorrow.
- RO1 + Amp (clones #1,2,3 in LB and M9+AA+thiam)
- RO2 + Amp (clones #4,5,10 in LB and M9+AA+thiam)
- RO1 + BB + Amp/Kana : is being transformed
- RO2 + BB + Amp/Kana (clones #1,3,4 in LB and M9+AA+thiam)
- LacI-RFP
Transformation
- RO1 #1 + BB1
- RO1 #2 + BB5
- RO1 #3 + BB3
Each time we want 10 ul of DNA, but 50% massic. A little calculation that could be useful :
- y = 10a / (a+b)
- x = 10 - y
With : a = concentration of RO1 in ng/ul
b = concentration of BB in ng/ul
x = the quantity of RO1 in ul to put
y = the quantity of BB in ul to put
Because : x + y = 10 ul, x*a = c = y*b
Ligation
The four BB (1,3,5,6) have been purified. The ligation run overnight at 4°C.
People in the lab
Mélanie, Caroline, Basile, Nicolas